Sequential hydrolyses of cheese whey proteins using trypsin, chymotrypsin and carboxypeptidase A (CPA) immobilized on agarose-glyoxyl were studied in this work. Phenylalanine (Phe) was removed from the hydrolysates by the action of CPA, thus providing an adequate source of proteins for phenylketonuria (PKU) patients. The role of the first two pre-hydrolyses on the yield of Phe by CPA was investigated: whey proteins were sequentially hydrolyzed to different degrees of hydrolysis with trypsin and chymotrypsin employing different enzyme/substrate ratios. The analyses indicate that a more suitable substrate for CPA is obtained when only chymotrypsin hydrolyzes whey. Using an ad-hoc approach, an empirical model for the apparent reaction rate was developed, which allows the prediction of the concentration of "hydrolyzed peptide bonds" (P) during the proteolysis with immobilized chymotrypsin. This model fitted the experimental data very well, while the Michaelis-Menten-type equation failed to represent the time-evolution of P in batch assays. (C) 2008 Elsevier Ltd. All rights reserved.